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