The tomato fruits during different stages of ripening have been extensively characterized for nutritionally important bioactives; however, changes in fatty acid composition are not available. Thus, in the present study, changes in fatty acid, along with carotenoid and α-tocopherol, were studied during the six stages of ripening. Fruits were harvested at the green, breaker, turning, pink, light red, and red stages, which occurred at means of 30, 35, 40, 46, 50, and 55 days after anthesis (DAE), respectively. During the ripening process, profiles of all the metabolites altered significantly (p < 0.05). All-E-lycopene content increased from the breaker (0.21 μg/g FW) to the red stage (30.6 μg/g FW), while all-E-lutein was slightly increased during initial stages of ripening and then decreased significantly, with the highest (4.15 μg/g FW) in the fruits of the pink stage. Furthermore, the contents of α-tocopherol increased during ripening, and its increase was highest between light red to the red stages. In all the ripening stages, linoleic acid (C18:2n6c) was found in the highest quantity (42.3-49.2%), followed by oleic (C18:1n9c; 20.1-26.6%) and palmitic acids (C16:0; 16.6-17.7%). With fruit ripening, the ratio of polyunsaturated fatty acids and saturated fatty acids (PUFAs:SFAs) was increased significantly from 1.89 (green) to 2.19 (red). Interestingly, the oleic acid proportions correlated inversely with linoleic (r = -0.450) and α-linolenic acid (r = -0.904), during all the stages of ripening. The highest and lowest contents of oleic acid and linoleic acid (26.7 and 42.3%, respectively) were recorded in the fruits of stage 3 (turning). In conclusion, ripening in tomatoes is accompanied by significant increases in carotenoids and α-tocopherol, as well as by concomitant increases in PUFAs.