ER-2 is a new investigational quinoline derivative synthesized by the Department of Organic Chemistry, University of Chennai, India, with significant activity against gram-positive and gram-negative organisms. Mycoplasma pneumoniae is a major cause of upper and lower respiratory tract infections across all age groups. This study compared the in vitro antibacterial activity of ER-2 against clinically relevant mycoplasma species—61 M. pneumoniae isolates from pneumonia clinical specimens and 40 M. hominis isolates from urogenital specimens (including 10 tetracycline-resistant strains)—with that of known antimicrobial agents (moxifloxacin, ciprofloxacin, levofloxacin, gatifloxacin, gemifloxacin, azithromycin, doxycycline, tetracycline, clindamycin). The agar dilution method was used to determine minimum inhibitory concentrations (MICs), with reference strains M. pneumoniae ATCC 29342 and M. hominis ATCC 43521; minimum bactericidal concentrations (MBCs) were tested for 5 M. pneumoniae isolates. Results showed ER-2 had excellent activity against M. pneumoniae, with MIC50 0.001 μg/ml and MIC90 0.016 μg/ml—equivalent to azithromycin and the lowest among tested drugs. MBCs (MBC50 0.001 μg/ml, MBC90 0.016 μg/ml) confirmed bactericidal activity against all M. pneumoniae isolates. M. hominis was highly susceptible to ER-2 (MIC90 0.06 μg/ml). The study indicates ER-2 is active in vitro against clinically important mycoplasmal species, with activity comparable to azithromycin and superior to tested fluoroquinolones. ER-2 shows promise for treating mycoplasma-related respiratory and urogenital infections, but further research on its toxicity and pharmacokinetics is needed.