This study aimed to investigate if canine staphylococci isolates could develop tolerance to chlorhexidine digluconate after long-term exposure. Staphylococci (Staphylococcus pseudintermedius and coagulase-negative staphylococci, methicillin-susceptible and methicillin-resistant), with and without genotypic chlorhexidine resistance, were investigated for phenotypic chlorhexidine tolerance by determining the MBC (minimal bactericidal concentration) at various time points. The testing was performed as follows: determination of MBC for 30 minutes and 24 hours (MBC 24h-1); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1; determination of MBC for 24 h after the first week (MBC 24h-2); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-2 and rest in Mueller–Hinton broth for seven days; determination of MBC for 24 h after the second week (MBC 24h-3). The MBC for 30 minutes ranged between 16–32 µg/ml compared to the MBC 24h-1 which was between 1–8 µg/ml. The MBCs for 24 h dropped from 8 µg/ml to 0.5 µg/ml for isolate 1, from 2 µg/ml to1 µg/ml for isolate 6 and from 2 µg/ml to 0.5 µg/ml for isolate 8 after being exposed for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1. For one CoNS (coagulase-negative staphylococci), the MBC 24h-2 increased four times from 1 µg/ml (MBC 24h-1) to 4 µg/ml and dropped again to 1 µg/ml after the second week. These results suggest that continuous exposure to chlorhexidine could lead to the selection of chlorhexidine-tolerant staphylococci that could withstand concentrations used during routine decolonisation procedures.