Introduction Increasing rates of antimicrobial resistance in Neisseria gonorrhoeae cause problems for treating gonorrhoea. Aim This observational study aimed to describe isolates from all patients found infected with N. gonorrhoeae, in Barcelona, Spain, between 2013 and 2017, and with available antimicrobial susceptibility data. Methods Minimum inhibitory concentrations (MICs) of penicillin (PEN), cefixime (CFM), ceftriaxone (CRO), azithromycin (AZM), ciprofloxacin (CIP), spectinomycin (SPT), fosfomycin (FOF) and gentamicin (GEN) were determined by E-test. Susceptibility was assessed using clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing. Time trends for PEN, CFM, AZM and CIP were investigated using logistic regression. Results Of 1,979 patients with infection (2,036 isolates), 1,888 (95.4%) were men. Patient median age was 32 years. The proportions of isolates resistant to extended-spectrum cephalosporins were low, with 0.3% (5/1,982) resistant to CRO and 4.9% (98/1,985) to CFM. AZM resistance prevalence was 2.7% (52/1,981), including 16 isolates detected in 2016 and 2017, with high-level resistance. For CIP, 51.3% (1,018/1,986) of isolates were resistant, and for PEN, 20.1% (399/1,985). All isolates were susceptible to SPT. MIC₅₀ and MIC₉₀ values of GEN were 4 and 6 mg/L and of FOF 12 and 24 mg/L, respectively. Between 2013 and 2017, PEN and CFM resistance rates each decreased from 28.1% (92/327) to 12.2% (70/572) and from 8.3% (27/327) to 4.4% (25/572) (p ≤ 0.0073). In contrast, AZM resistance prevalence appeared to increase from 1.5% in 2014 (5/340) to 3.0% (17/572) in 2017. No trend was identified for CIP. Conclusion Antimicrobial susceptibility surveillance is important to timely detect new phenotypes and trends.