Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are important pathogens affecting feline hosts worldwide, compromising the health, well-being, quality of life, and lifespan of infected cats. This study explores the prevalence of these viral agents among shelter cats in Portugal, aiming to unravel their complexities and implications. The prevalence of FIV and FeLV was assessed by collecting blood samples (n = 326) from cats during medical routine check-ups at shelter admission and testing them with an immunochromatographic kit. The overall seroprevalence of FIV and FeLV was 15.3% (50/326, 95% confidence interval [CI]: 11.6–19.7). The proportion of cats positive to FIV, FeLV, and both FIV and FeLV was 9.8% (32/326, 95% CI: 6.8-13.6), 2.1% (7/326, 95% CI: 0.9-4.4) and 3.4% (11/326, 95% CI: 1.7-6.0), respectively. The prevalence of FIV and both FeLV and FIV appeared to be higher in the adult population. No statistical association was found between sex, breed, hair, or municipality, and FeLV, FIV and both FeLV and FIV prevalence. This is the first report on retroviral prevalence in shelter cats in Portugal, which provides important information regarding FIV and FeLV among stray cats in Portugal, highlighting the need for surveillance of those viruses, improved compliance with vaccination programmes, and vigilant management strategies to limit and control these viruses in the felinepopulation.