Mycoplasma bovis is a major cause of pneumonia, arthritis, and mastitis in cattle and can lead to significant economic losses. Antimicrobial resistance is a concern and further limits the already short list of drugs effective against mycoplasmas. The objective of this study was to examine changes in in vitro minimum inhibitory concentrations (MICs) of antimicrobials of aminoglycoside, fluoroquinolone, lincosamide, macrolide, pleuromutilin, phenicol, and tetracycline classes for 210 M. bovis isolates collected from 1978 to 2009. The MIC(50) values of the various antimicrobials were also compared. The MIC(50) levels for enrofloxacin and danofloxacin remained low (0.25 μg/mL) across all 3 decades. MIC(50) levels for tetracyclines, tilmicosin, and tylosin tartrate were low in the 1980s, then increased in the 1990s and remained high. In the 1980s, MIC(50) levels were low for clindamycin, spectinomycin, and tulathromycin, increased in the 1990s to 8 μg/mL (clindamycin) and 32 μg/mL (spectinomycin and tulathromycin), then decreased again in the 2000s. Members of the fluoroquinolone class of antimicrobials had the lowest MIC(50) levels across all 3 decades, which suggests in vitro susceptibility of M. bovis to this class of antimicrobials. Statistically significant associations were observed between MIC values for chlortetracycline, oxytetracycline, tylosin tartrate, and tilmicosin; between clindamycin, tulathromycin, spectinomycin, and tiamulin; and between tylosin tartrate and clindamycin. Changes in MIC levels of various antimicrobials over time show the importance of monitoring the susceptibility of mycoplasmas to antimicrobials. The number of antimicrobials that showed elevated MIC(50) levels, and therefore possibly reduced in vitro effectiveness against M. bovis, supports initiatives that promote prudent use of antimicrobials in agriculture.