Data concerning Neisseria gonorrhoeae infections in Italy are scarce, and there is little information on the phenotypic and genotypic characteristics of the circulating strains. In this study, 326 isolates collected from 397 patients, with or without concurrent human immunodeficiency virus (HIV) infection, were cultured and characterized by serovar and antimicrobial susceptibility to five antimicrobials. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was also performed for strain characterization and to identify a transmission network. Gonococcal infection was diagnosed in 364 males and 33 females, 296 of whom were Italian and 96 of whom were foreigners (nationality was unknown in five cases). Among the 364 males, 197 were heterosexual, and the median age was 31 years. Approximately 8.3% of all the investigated patients were HIV-1-positive. The isolates were assigned to three different serovars (IA, IB, IA/IB), IB being the most frequently encountered. A significant rate of resistant gonococci was also observed; 34%, 25.5% and 19.1% of ciprofloxacin-resistant, penicillin-resistant and tetracycline-resistant phenotypes, respectively, were detected, and 10.2% of strains were multidrug-resistant. Together with the presence of different sequence types (STs), identified by NG-MAST, a multidrug-resistant cluster, ST661, was detected in a heterosexual network in a precise geographical area of the country. In particular, all strains belonging to ST661 showed identical profiles according to pulsed-field gel electrophoresis (PFGE), all were serotype IB, and all were resistant to penicillin, ciprofloxacin and tetracycline.