Mycoplasma spp. are difficult to culture because of their fastidious growth requirements such as atmospheric carbon dioxide (CO₂) levels exceeding 3%. Recommendations for culture CO₂ concentrations are varied and not empirically derived. The objective was to determine if bovine mycoplasma isolates differ in growth measures when incubated in CO₂ concentrations of 10% and 5% or in candle jars (2.7 ± 0.2% CO₂). Growth at these CO₂ concentrations was evaluated using isolates of the 3 most common mycoplasma mastitis pathogens, M. bovigenitalium (n = 5), M. bovis (n = 19), and M. californicum (n = 18). Isolates were standardized to OD₅₄₀ of 0.20, roughly 10⁸ cfu/mL, and serially diluted in pasteurized whole milk to achieve final concentrations of 10² and 10⁶ cfu/mL. One hundred microliters of each dilution was spread onto the surface of a modified Hayflick’s agar plate to enumerate colony growth on d 3, 5, and 7 of incubation. SAS 9.3 GLM was used to evaluate fixed effects of CO₂ treatment (2.7, 5, or 10% CO₂), species, and day (3, 5, or 7 d) and their interactions on recorded colony counts. Carbon dioxide concentration and day were not associated with overall mycoplasma growth differences but species differences were identified (P < 0.0001). Species GLM models assessed fixed effects of CO₂ treatment, isolate, and day and their interactions on recorded colony counts isolate growth differences. For M. bovis, CO₂ concentration and day did not significantly affect growth, but growth differences among isolates were identified (P < 0.0001). A significant interaction between isolate and CO₂ concentration was found for M. californicum and M. bovigenitalium (P < 0.0001 and P = 0.007, respectively). Growth after 7 d incubation was significantly greater for M. bovigenitalium and M. californicum (P < 0.0001). These findings are being confirmed on field isolates received from a commercial veterinary diagnostic laboratory. The results suggest that the range of suitable CO₂ culture conditions and incubation times for the common mastitis-causing Mycoplasma spp. is broader than currently practiced.