In recent years, there has been increasing interest in the study of extremophilic microorganisms, which include halophiles and halotolerants. These microorganisms, able to survive and thrive optimally in a wide range of environmental extremes, are polyextremophiles. In this context, one of the main reasons for studying them is to understand their adaptive mechanisms to stress caused by extreme living conditions. The present study investigated the halotolerance and metabolic responses of five Eurotium species (E. amstelodami, E. chevalieri, E. cristatum, E. montevidense, and E. proliferans) under varying sodium chloride (NaCl) concentrations (0–25%). Fungal growth, medium pH, pigment production (visible light absorbance at 512 nm), fluorescence (254 and 366 nm), organic matter, total nitrogen content, and antimicrobial activity against bacterial (Bacillus subtilis, Escherichia coli) and fungal (Candida tropicalis, Candida albicans) strains were evaluated. All isolates exhibited halotolerant behavior, with maximal growth, organic matter, nitrogen content, fluorescence, and pigment accumulation at moderate salinity (15% NaCl). Antimicrobial activity was strongest at low to moderate NaCl concentrations (0–10%) and absent at ≥15% NaCl, reflecting the inhibitory effect of extreme salinity on secondary metabolite production. The pH of the culture media varied with salinity, generally decreasing under moderate salt stress and increasing at the highest NaCl levels. These patterns indicate that mild osmotic stress promotes both primary and secondary metabolism, whereas severe salinity suppresses growth and bioactive compound synthesis. The study highlights the ecological adaptability of Eurotium species to saline environments and their potential as sources of halotolerant enzymes, pigments, and antimicrobial compounds