The susceptibility of various animal species to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been studied extensively. Cats have garnered significant concern due to their high susceptibility and proximity to humans. This study aimed to evaluate the susceptibility and antibody response in house cats exposed to SARS-CoV-2 when human infection was spreading in the Lisbon area during the 2019-2021 period. A total of 733 serum samples were collected and characterized from cats admitted to the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine at the University of Lisbon (HEV-FMV-ULisboa). All samples were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin M (IgM) and immunoglobulin G (IgG) anti-SARS-CoV-2 whole Spike and receptor-binding domain (RBD) proteins from the Wuhan-Hu-1 isolate and 14.7% (108/733) tested positive, suggesting exposure to the human virus. Surrogate virus neutralization test (sVNT) against the Wuhan-Hu-1 isolate showed that 20.4% of ELISA positive samples (22/108) harbored neutralizing antibodies against the virus. The 22 most promising serum samples were retested using ELISA and sVNT against Alpha, Delta, and Omicron SARS-CoV-2 variants. Notably, these samples exhibited antibodies that were capable of recognizing and neutralizing these variants. Subsequent neutralization assays confirmed that the serum samples effectively inhibited the infection process of Wuhan-Hu-1 D614G, Delta, and Omicron SARS-CoV-2 pseudotyped viruses. Our findings indicate that cats were exposed to SARS-CoV-2 infection during the pandemic period and generated highly effective and broadly neutralizing antibodies against the virus. Although cats have not been demonstrated to significantly contribute to the spread of SARS-CoV-2, their high susceptibility to asymptomatic infection underscores the importance of investment in preventive surveillance measures. In summary, our study reinforces the notion that cats naturally infected with SARS-CoV-2 represent a valuable anthroponotic disease model in house settings and might be a potential source for the development of antibodies against SARS-CoV-2 in tackling future outbreaks with a One Heath perspective.