Background: Polyunsaturated fatty acids (PUFAs), particularly Omega-3 (ω-3) and Omega-6 (ω-6) PUFAs, may exert neuroprotective effects via the endocannabinoid system (ECS) and are promoted as brain health supplements. However, despite their potential role in endocannabinoid biosynthesis, the impact of PUFAs on ion channels such as TRPV1 and TRPA1, which are modulated by endocannabinoids, remains incompletely understood. Furthermore, the potential in vitro actions of ω-6 to ω-3 PUFA combined in the ratios available in supplements remains uncertain. Therefore, the objective of this study is to evaluate the functional activity of individual PUFAs, their combination in a specific ratio, and their endocannabinoid-related derivatives on TRPV1 and TRPA1 ion channels. Methodology: We employed a fluorescent Ca-sensitive dye in HEK-293 Flp-In T-REx cells expressing human TRPV1, TRPA1, or an empty vector to measure changes in intracellular calcium concentration ([Ca2+]i). Results: Capsaicin and certain PUFA derivatives such as DHEA, γ-LEA and AEA stimulate TRPV1 activity directly, whereas EPA, DHA, γ-LA, and their 9:3:1 ratio triggered TRPV1 response via a mechanism dependent on prior exposure to phorbol ester. Similarly, cinnamaldehyde and selected PUFA derivatives such as EPEA, DHEA, γ-LEA, 2-AG, 2-AG ether and AEA triggered TRPA1 response, with EPA, DHA, γ-LA, and the 9:3:1 ratio showing significant effects at higher concentrations. Conclusions: PUFAs alone and their combined form in 9:3:1 ratio stimulate TRPA1 activity, whereas their metabolites trigger both TRPV1 and TRPA1 response. These findings suggest new avenues to explore for research into potential mechanisms underlying the neurological benefits of PUFAs and their metabolites.