Control programs for contagious agalactia (CA) involve monitoring milk samples to detect this disease. This study was designed to establish the effects of the preservatives generally used in dairy laboratories and storage temperature on the viability of Mycoplasma (M.) agalactiae (Ma) and M. mycoides subsp. capri (Mmc) in goat milk samples. In total, 1440 determinations were conducted for each mycoplasma species in milk samples subjected to different storage temperatures (refrigeration at 4°C or freezing at -20°C), preservation strategies (no preservative, NP; azidiol, AZ; or bronopol, BR) and storage times at each temperature (0, 2, 4, 6, 8, 10 and 24h at 4°C and 48h, 1 week, 2 weeks and 4 weeks at -20°C). Our findings reveal the similar viability of Mmc in milk samples stored at 4°C for 24h under the three preservation conditions examined. In contrast, the isolation of Ma in refrigerated milk samples was compromised by the presence of BR, and in smaller measure by the treatments AZ and NP. Freezing milk samples considerably reduced the viability of both mycoplasmas. Given the different sensitivity of the two mycoplasma species to BR, refrigerated milk samples treated with AZ could be used to detect infections caused by both species through culture-based methods.