Purpose The aim of this study was to investigate homophobic attitudes in three European countries: Italy, Albania, and Ukraine. One thousand and forty-eight students were recruited in Italian ( n  = 766), Albanian ( n  = 180), and Ukrainian ( n  = 102) university centers. Methods A socio-demographic questionnaire and Homophobia Scale (HS) were administered by our staff. Results Cross-cultural and significant differences among Italian, Albanian, and Ukrainian students were found on the Homophobia Scale (HS; Italy: mean = 22.26 ± 16.73; Albania: mean = 38.15 ± 17.28; Ukraine: mean = 59.18 ± 16.23). The analysis of socio-demographic characteristics revealed that the male gender emerged as main predictor of homophobic attitude in all the three countries, although also a conservative political orientation and the religious belief predict higher homophobia levels in Italy and Albania, particularly. Conclusions This study revealed that in these European countries assessed, attitudes toward homosexuality are different. Ukrainians display higher levels of homophobia than Albanians and Italians, confirming the central role of cultural differences in homophobic attitudes. Nevertheless, some socio-demographic aspects such as identification as male have a similar influence on homophobic attitudes in all assessed populations.