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Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis . Despite a wide range of carbon dioxide (CO 2 ) supplementation reported in literature, specific impacts of CO 2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO 2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis , were incubated at 37°C in triplicate and three dilution ranges under three CO 2 conditions: ambient air or 5% CO 2 or 10% CO 2 . Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log 10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO 2 , with less variation observed in ambient air compared with 5% or 10% CO 2 . However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO 2 , or 10% CO 2 . Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7-10 days of culture, even in the absence of supplemental CO 2 . Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis , we recommend expanding CO 2 concentration ranges in M. bovis culture from 10% CO 2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO 2 ., Importance: Current Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO 2 ) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis . This study indicates that the suitable range for CO 2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7-10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO 2 , or 10% CO 2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.