The presence of mycotoxins and other toxic metabolites in hops (Humulus lupulus L.) was assessed for the first time. In total, 62 hop samples were sampled in craft breweries, and analyzed by a multi-toxin LS-MS/MS method. The study collected samples from craft breweries in all of the Croatian counties and statistically compared the results. Based on previous reports on Alternaria spp. and Fusarium spp. contamination of hops, the study confirmed the contamination of hops with these toxins. Alternaria toxins, particularly tenuazonic acid, were found in all tested samples, while Fusarium toxins, including deoxynivalenol, were present in 98% of samples. However, no Aspergillus or Penicillium metabolites were detected, indicating proper storage conditions. In addition to the Alternaria and Fusarium toxins, abscisic acid, a drought stress indicator in hops, was also detected, as well as several unspecific metabolites. The findings suggest the need for monitoring, risk assessment, and potential regulation of Alternaria and Fusarium toxins in hops to ensure the safety of hop usage in the brewing and pharmaceutical industries. Also, four local wild varieties were tested, with similar results to the commercial varieties for toxin contamination, but the statistically significant regional differences in toxin occurrence highlight the importance and need for targeted monitoring.