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Bacterial biofilms are involved in the aggravation and recurrence of clinical mastitis in dairy herds. Several factors such as pH, temperature, concentration of O2 and glucose can affect their induction and growth rates. In this study, biofilm production was demonstrated by 27 Escherichia coli strains isolated from bovine mastitis at different pH values depending on the availability of glucose, mimicking conditions found in mammary glands affected by the disease. Biofilm formation was analyzed by spectrophotometric analysis in microtiter plate with 16 different culture media and by scanning electron microscopy. Biofilm formation was greater in isolates cultured under conditions associated with low glucose availability (0.5% or 1.5%) and with either an acidic (5.5) or alkaline (8.5) pH, compared to conditions associated with high glucose availability (2.5% or 3.5%) and near-neutral pH (6.5 or 7.5). Results indicate possible favoring of biofilm production in the later stages of the infectious process caused by E. coli, when the gland environment is less propitious to bacterial growth due to the stress conditions mentioned above; contrasting with the environment of the healthy mammary gland, in which there is no limitation on nutrients or conditions of particular alkalinity or acidity. Thus, knowledge of the stage in which is the infection and environmental conditions of the mammary gland that cause increased production of biofilms is of paramount importance to guide the most appropriate control strategies to prevent relapse after treatment of bovine mastitis, an economically important disease in dairy cattle worldwide.