Remediation of hydrocarbon-contaminated groundwater is challenging due to the large volume of contaminated water, restricted aquifer access, and the recalcitrance of hydrocarbons. This study evaluates chemically-based surfactants (A and B, comprised of alcohols, esters and distillates from petroleum) and biosurfactants (C and BS, containing enzymes and microbial-derived surfactants) to enhance petroleum-hydrocarbon remediation. Surfactants/biosurfactants were evaluated under environmental conditions mimicking Arabic Region groundwater. The influence of factors like surfactant to crude oil ratios (S:O), temperature, and dissolved oxygen on the remediation performance was analyzed. Among studied surfactants, C and BS demonstrated the best performance. Multi-spike of C achieved the highest reduction of crude oil layer thickness (41.9%) and total petroleum-hydrocarbons (TPH) removal (20.6%). However, since dispersion mechanisms drove removal, it led to increased turbidity (3.5 NTU) and toxicity (61.6% biofluorescence-inhibition) in the water phase. In contrast, BS with a similar performance in terms of crude oil layer reduction (38.5%) and TPH removal (17.8%) resulted in lower levels of turbidity (1.8 NTU) and toxicity (26.8% biofluorescence-inhibition). This is attributed to the removal via both dispersion and biodegradation. BS-biodegradation was associated to a high relative abundance of Pseudomonas spp. with abilities to degrade low molecular and polyaromatic hydrocarbons (i.e., BTEX and PAHs). Results demonstrated BS is the most sustainable product to enhance hydrocarbons removal. Significant influence in the remediation process was observed at optimal S:O ratio (C-1:5 BS-1:10) and high temperature of 35 °C. Our findings elucidated the mechanisms involved in groundwater remediation via surfactants/biosurfactants, and provide optimal parameters for its application in-situ.