Sialic acids in the structural matrix of biofilms developing in engineered water systems constitute a potential target in the battle against biofouling. This report focuses specifically on the presence of sialic acids as part of the extracellular polymeric substances (EPS) of biofilms forming in cooling towers and the potential effect of nutrient starvation on sialic acid presence and abundance. Two cooling water compositions were compared in parallel pilot-scale cooling towers, one poor in nutrients and one enriched in nutrients. Fresh deposits from the two cooling towers were collected after a five-week operation period. EPS extractions and analyses by Fourier transform infrared spectroscopy (FTIR) and high-resolution mass spectrometry (MS), along with 16S rRNA gene amplicon sequencing were performed. The results of MS analyses showed the presence of pseudaminic/legionaminic acids (Pse/Leg) and 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) in both biofilm EPS samples. FTIR measurements showed the characteristic vibration of sialic acid-like compounds ν(C=O)OH in the nutrient poor sample exclusively. Our findings, combined with other recent studies, suggest that bacterial sialic acids are common compounds in environmental biofilms. Additionally, the conservation of sialic acid production pathways under nutrient starvation highlights their importance as constituents of the EPS. Further in-depth studies are necessary to understand the role of sialic acids in the structural cohesion and protection of environmental biofilm layer.