Membrane progesterone receptors (mPRs) are members of the progestin and adipoQ (PAQR) receptor family that are stimulated by endogenous steroids to initiate rapid intracellular signaling through a nongenomic pathway. Previously, water-soluble compounds with mPRα binding activity from the marine algae Padina arborescens were fractionated by HPLC. Nuclear magnetic resonance spectroscopy revealed that the major component of the HPLC fraction was 2-hydroxypentanoic acid (2-HPA). In this study, the physiological activity of 2-HPA and its analogues was investigated using in vitro goldfish and in vivo zebrafish oocyte maturation and ovulation assays. Only 2-HPA showed inhibitory activity on oocyte maturation and ovulation of fish oocytes. The inhibitory activity of 2-HPA was compared between S- and R-type 2-HPA. The results showed that both types had the same level of activity. Furthermore, the interaction of 2-HPA with mPRα was analyzed by binding assay. 2-HPAs showed a substantial competitive binding affinity for the human membrane progesterone receptor α (hmPRα) in the graphene quantum dots (GQDs)-hmPRα binding assay. In contrast, synthetic structural analogues of 2-HPA showed no competitive binding activity. These results indicate that 2-HPA is a novel mPRα antagonist, and its chemical structure is highly restricted to show its activity.