Low back pain is a serious threat to human health and the illness jeopardizes the human workforce and pressurizes the health system in the community. Low back pain might be related to piriformis syndrome (PS), which is a disorder presented as muscular spasm and hypertrophy that is strongly associated with piriformis thickness. Nevertheless, the relationship between piriformis thickness and morphological and functional changes of the gluteal muscles in PS remains unclear. This study aimed to investigate the association between the thickness, strength, and activation of piriformis and gluteus muscles (maximus and medius) among low back pain (LBP) patients with and without PS. This is a case-control study conducted at HSNZ and UiTM from 2019–2020. A total number of 91 participants (LBP + PS (n= 36), LBP − PS (n= 24), and healthy (n= 31)) were recruited in this study. Negative radiography, specific symptoms, and a positive PS test were applied for PS diagnoses. The thickness, strength, and activation of piriformis and gluteus muscles were measured using ultrasonography (USG) and a surface electromyogram, respectively. Resultantly, the one-way ANOVA test demonstrated no significant difference in piriformis thickness between LBP + PS and LBP − PS (p> 0.01). Piriformis thickness was inversely correlated with gluteus maximus strength (r = −0.4,p< 0.05) and positively correlated with gluteus medius activation (r = 0.48,p< 0.01) in LBP + PS. Stepwise linear regression for LBP + PS revealed a significant association between piriformis thickness and gluteus maximus strength (R = −0.34, accounted for 11% of the variance) and gluteus medius activation in prone lying with the hip in an externally rotated, abducted, and extended (ERABEX) position (R = 0.43, accounted for 23% of the variance). With the adjustment of age and gender, piriformis thickness, gluteus maximus strength, and gluteus medius activation in prone lying with hip ERABEX demonstrated a significant association, but no independent effect of age and gender was detected within the range. Meanwhile, a significant association between piriformis thickness and gluteus maximus thickness was observed (R = 0.44, accounted for 19% of the variance) in the LBP − PS group. These findings may assist to elucidate the actions and functions of piriformis and gluteus muscle in LBP with and without PS.