ABSTRACT: Antimicrobial resistance (AMR) is an urgent and complex problem worldwide, exacerbated by the frequently inappropriate use of antibiotics. The purpose of this study was to survey the levels of knowledge and awareness about antibiotic use and stewardship, among human and veterinary health professionals or students in Portugal, and the associations between antibiotic knowledge factors and socio-professional groups. In cross-sectional survey design, a total of 449 online structured questionnaires were completed in 2018-2019. The statistical analysis was performed dividing the respondents into four groups, A (undergraduate students), B (PhD students and researchers), C (lecturers), and D (technicians and other occupation). Among all respondents, 17% (n = 75) revealed some gap in knowledge about antibiotic resistance and the antibiotics that should be administered for different infection types (bacterial, viral, or fungal). Of the 159 pet owners among the respondents, only half had administered antibiotics to their animal and 64% (n = 102) knew that veterinary prescription is mandatory when administering antibiotics to animals. All groups statistically agreed that the AMR is a major public health problem and the antibiotics should be administrated for bacterial infections and used until the whole pack has been finished (p = 0.00). As expected, only groups B and C demonstrated a higher level of knowledge to recognize the antibiotic name and their active ingredient than undergraduate students (p = 0.00). About the antibiotic use on pets, only group B was statistically significant to no used antibiotics on their pets (p = 0.00). However, groups A, C, and D were statistically significant for the knowledge about the mandatory veterinarian prescription and groups C and D were significantly statistics for fully aware of the transmission of bacteria between animals and humans. In conclusion, in matters related to AMR, the behavior, education, and training of the general public and health professionals, including those who prescribe antibiotics for humans and animals, need to be improved.