Background: Lateral instability of the ankle is one of the most common causes of musculoskeletal ankle injuries. The peroneus longus (PL) and peroneus brevis (PB) contribute to ankle stability. In early rehabilitation, isometric exercises have been selected for improvement of ankle stability. To effectively train the peroneal muscles during eversion, it is important to consider ankle and body posture. Objects: This study aimed to compare activation of the PL, PB, and biceps femoris (BF) muscles during eversion in different ankle postures (neutral [N], plantarflexed [PF]) and body postures (sitting and side-lying). Methods: Thirty healthy individuals with no history of lateral ankle sprains within the last 6 months were included in the study. Maximal isometric strength of eversion and muscle activation were measured simultaneously. Muscle activation at submaximal eversion was divided by the highest value obtained from maximal isometric eversion among the four postures (percent maximal voluntary isometric contraction [%MVIC]). To examine the differences in muscle activation depending on posture, a 2 × 2 repeated measures analysis of variance (ANOVA) was conducted. Results: There were significant interaction effects of ankle and body postures on PL muscle activation and evertor strength (p < 0.05). The PL muscle activation showed a significantly greater difference in the side-lying and PF conditions than in the sitting and N conditions (p < 0.05). Evertor strength was greater in the N compared to the PF condition regardless of body posture (p < 0.05). In the case of PB and BF muscle activation, only the main effects of ankle and body posture were observed (p < 0.05). Conclusion: Among the four postures, the side-lying-PF posture produced the highest muscle activation. The side-lying-PF posture may be preferred for effective peroneal muscle exercises, even when considering the BF muscle.