Traces of COVID-19 were first detected in Shepparton wastewater on Wednesday, August 11, prompting a push for higher testing rates in the Greater Shepparton area.
But Chief Health Officer Brett Sutton said wastewater had since tested positive for a second time, and urged anyone with the "slightest of symptoms" in Shepparton, Kialla and Orrvale to get tested.
"It's not definitive of there being a case there, but I want people to consider if they are symptomatic to come forward for testing," Mr Sutton said.
"If you live or work in these areas, you don't have to reside there, if you are moving into those areas, especially as an essential worker, please get tested."
It comes with 24 new locally acquired cases of COVID-19 reported in Victoria on Tuesday including 10 cases that were in the community while infectious.
Twenty-one of today's new cases are linked to ongoing outbreaks and three remain under investigation by health authorities, with growing concern over mystery cases bubbling in the St Kilda region.
Three cases are linked to Al-Taqwa College, nine are linked to Glenroy West Primary School and four are linked to the St Kilda East community outbreak including social contacts who breached quarantine to attend a private gathering.
Two new cases are linked to a Carlton housing tower and three are linked to the Newport community outbreak.
There was one new case detected in hotel quarantine.
From today, Melbourne has entered harsher lockdown restrictions including a 9pm to 5am curfew and the closure of playgrounds, basketball courts and outdoor exercise equipment.
Authorised workers will require a permit to travel.
Regional Victoria remains under the same restrictions put in place before the latest Delta outbreak began but Melbourne's stay-at-home restrictions have been extended until September 2 in a bid to curb ongoing chains of unknown transmission across the city.
There have been no new exposure sites listed in regional Victoria since the latest outbreak began.
Today's new cases were detected from 31,519 test results, with 25,742 vaccine doses administered across state-run sites.
GV Health COVID-19 vaccine program executive lead Alicia Cunningham said COVID-19 tests had doubled at GV Health since the positive wastewater test in Shepparton was detected.
"Prior to the wastewater detection, GV Health was conducting approximately 100 COVID-19 tests per day," she said.
"Since then, we've been testing around 200 people per day. This is very encouraging as it shows that our community is doing the right thing to get tested if they have COVID-19 symptoms, no matter how mild."
Premier Daniel Andrews urged for higher testing numbers in the coming days and said while vaccinations were key to emerging out of the pandemic, test results were crucial to suppressing the Delta variant "in the short term".
There are now 227 active COVID-19 cases across the state.
Further north, 452 new locally acquired cases of COVID-19 were detected in NSW overnight, with investigations continuing into where "a number of cases" acquired the virus.
There has been one further death - an unvaccinated woman in her 70s.
More to come.