The use of ethephon, designed to stimulate nut detachment, initiates the release of ethylene, a well-established regulator of postharvest shelf-life in various agricultural products. This study aimed to assess the impact of ethephon application on individual fatty acids and dietary indicators in two macadamia nut cultivars, namely ‘788’ and ‘Beaumont,’ during postharvest storage. Nuts that naturally abscised and those detached through ethephon treatment were divided into two groups: the ethylene-treated group (ED) and the control group (CD). Nuts were stored at 25°C and sampled at 0, 36, and 72 days for fatty profile analysis. Our findings indicated a significant increase in stearic acid content in ED nuts (24,622 µg/g) compared to CD nuts (16,764 µg/g) at the end of the storage period for the ‘Beaumont’ cultivar. Additionally, unsaturated fatty acids (USFAs), such as eicosatrienoic acid + erucic acid (C20:3n3+C22:1) and eicosatrienoic acid + alpha-linolenic acid (C20:1 + C18:3n3), were notably reduced. Hierarchical clustering analysis revealed positive correlations between ethylene treatment and saturated fatty acids (SFAs) in both ‘Beaumont’ (0.78) and ‘788’ (0.80) cultivars. This also coincided with an increase in atherogenic indices, thrombogenic index, and saturation index and a decrease in the hypocholesterolemic/hypercholesterolemic ratio and arachidonic acid (C20:4n6) within the ED group of the ‘Beaumont’ cultivar, collectively potentially impacting nutritional quality negatively. Furthermore, our findings indicated that the PUFA:SFA ratio was higher in CD (0.51) compared to ED (0.45) on day 72 for the ‘Beaumont’ cultivar, revealing differences in fatty acid compositions between the two treatment groups. Conversely, for ‘788’, both ED and CD had a PUFA:SFA ratio below 0.45, indicating an increased risk of cardiovascular diseases. These results suggest that ethephon treatment increases SFA levels and reduces USFA levels in the ‘Beaumont’ cultivar, while the response to ethylene varies between the two cultivars. Thus, the study provides insight into the significant role of modifying ethephon treatment methods and careful cultivar selection in the attainment of optimal nutritional value and shelf-life of macadamia nuts.