At 2.30am on Saturday, a tram travelling from Luna Park in St Kilda to Malvern depot sold Melbourne's last Metcard as the switchover to myki became official.
Public transport users must now carry a myki on trains, trams and buses, or face fines for fare evasion.
A spokesman from the Transport Ticketing Authority told ABC radio that inspectors would be on their regular rounds and while they would not "capitalise" on the changeover, they would not accept not having a myki as an acceptable excuse for not carrying a valid ticket.
Metcard validating machines at train stations, which were introduced in 1996, are now bearing "out of service" stickers, and vending machines have been switched off ahead of their removal from stations.
The changeover means that tourists and other infrequent users of public transport must pay $6 for a myki card – $3 if they are a concession card holder or senior – prior to loading their fare on the card.
To help tourists, myki tourist packs will be sold at Melbourne Airport and on the Skybus. Bus drivers on some routes will carry a limited number of pre-loaded myki cards for passengers who find themselves caught short away from a myki retail outlet.
The controversial $1.5 billion ticket system has been plagued by cost overruns, technical failures, and its inability to offer cheap short-term tickets for tourists.
It also means that passengers are unable to buy a ticket on a tram, the first time this has happened since the tram network opened in 1885.
Myki cards can be bought and topped up at premium train stations, selected tram stops, and over 800 retail outlets such as 7-Elevens.
The changeover comes as public transport fares are set to increase on January 1. A full-day myki fare in zone 1 will increase from $6.56 to $7, while weekly tickets will increase by $2.20 to $35.