<p>The development of naturally occurring compounds is highly desirable for devastating food spoilage-causing fungi, controlling the rapid emergence of antibiotic-resistant pathogens, and enzyme-linked human melanogenesis. The primary aim of the current study was to identify natural sources with potential antimycotic, antibacterial, and antityrosinase activities as alternatives to synthetic compounds. Gas chromatography–mass spectrometry (GC-MS) analysis revealed that the major bio-constituents of chia seed oil (CFSO) were oleic acid (31.13%), stearic acid (19.36%), linolenic acid (13.68%), and palmitoleic acid (11.48%). The fixed oil displayed a significant inhibition (<i>P</i> &lt; 0.05) of mycelial growth in a dose-dependent manner. The highest antifungal inhibition percentage was recorded for <i>Penicillium expansum</i>. CFSO significantly reduced the growth of <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, and <i>Enterococcus faecalis</i>, whereas <i>Pseudomonas aeruginosa</i> was not affected at concentrations ranging from 12.5 to 25.0%. The most sensitive species (<i>S. aureus</i>) showed high ultrastructure fluctuations. Kinetic analyses showed that CFSO competitively inhibits the diphenolase activity of tyrosinase (IC₅₀ &lt; 220&#xa0;µg/ml), whereas its effect on monophenolase activity was weaker (IC₅₀ &gt; 500&#xa0;µg/ml). The docking analysis showed a strong interaction between an oxygen atom of the major fatty acids and the copper atoms (Cu⁴⁰⁰ and Cu⁴⁰¹) and the histidine residues (His²⁵⁹, His⁶¹, His²⁶³, His⁸⁵, His²⁹⁶, and His⁹⁴) in the tyrosinase binding pocket. This is probably the first study investigating the inhibition kinetics of chia oil on diphenolase activity of tyrosinase with computational studies. The active compounds of CFSO could be potentially utilized through antimicrobial therapy, food preservation, neoteric cosmetic formulations, and medicinal innovation.</p>