Objectives In recent years, resistance in Mycoplasma genitalium (MG) to first-line (azithromycin) and second-line (moxifloxacin) treatment has been increasingly reported worldwide, however, no data regarding the south of Spain are available.

Methods To determine resistance rates, MG-positive samples collected from June 2018 to June 2019 were analysed by sequencing the 23S rRNA and parC genes.

Results A total of 77 patients (24 men having sex with men (MSM), 30 heterosexual men and 23 women) were included. Resistance-associated mutations against macrolide and fluoroquinolones were found in 36.4% (95% CI 25.7% to 48.1%) and 9.1% (95% CI 3.7% to 17.8%) of the patients, respectively. Being MSM and having had another STI in the last year were significantly associated with macrolide-resistant MG infection, while no associations were found with resistance to fluoroquinolones.

Conclusions Testing for resistance to first-line and second-line drugs against MG should be recommended for the general population and mandatory for the MSM population. We suggest that empiric azithromycin use for STI management should be avoided.