The control of biodeteriogenic microorganisms is essential for the management of heritage sites. Many conventional biocides are no longer available because they have lost their efficacy or have been withdrawn from the market due to their danger to humans and the environment. Therefore, new effective and sustainable biocides are needed, such as plant extracts that could be a good alternative. In this study, essential oils (EOs) of Ocimum basilicum L., Cinnamomum verum Presl, Lavandula angustifolia Mill., Origanum vulgare L., Thymus vulgaris L. and Melaleuca alternifolia Maiden & Betche were tested as green biocides against microorganisms collected from biofilms in the hypogeum of the Colosseum (Rome, Italy). Biocidal screening was first carried out on phototrophic microorganisms grown on BG11 agar culture medium. The efficacy was assessed by measuring photosynthetic activity with a mini-PAM portable fluorometer, and by determining morphological changes or the absence of autofluorescence using light microscopy and confocal laser scanning microscopy. The most effective EOs against phototrophs were further tested to inhibit the growth of heterotrophic fungi and bacteria in order to identify those with a broad-spectrum action. The EOs of cinnamon, oregano and thyme at 5% concentration (v/v) were the most effective against the microorganisms isolated from the biofilms in the Colosseum. These EOs represent a green alternative to traditional chemical biocides due to their activity against a wide range of microorganisms and their complex composition which suggests the potential to reduce the risk of microbial resistance. KEY POINTS: Biofilms collected from the Colosseum hypogeum were characterized EOs tested as biocides against phototrophs and heterotrophs in Colosseum biofilms. Cinnamon, oregano, and thyme EOs show broad-spectrum action at 5% concentration.