Petroleum hydrocarbon contamination in aquatic systems poses persistent ecological and toxicological risks, necessitating efficient and sustainable remediation strategies. This study assessed the efficacy of an integrated nano-phytoremediation system combining Eichhornia crassipes and nanoscale zero-valent iron (nZVI) for removing crude petroleum oil from contaminated water. Experimental setups with 1000 ppm CPO included control, phytoremediation-only, nZVI-only, and hybrid treatments monitored over 28 days. Results revealed a clear hierarchy in total petroleum hydrocarbon removal efficiency, with hybrid systems achieving significantly greater reductions than standalone treatments. By week 4, TPH levels in the highest-dose hybrid system declined from an initial 5.10 ± 0.22 mg/L to 1.28 ± 0.10 mg/L, compared to 2.96 ± 0.18 mg/L for phytoremediation alone and 2.21 ± 0.15 mg/L for nZVI-only. This superior performance stems from synergistic mechanisms, including adsorption, reductive transformation, rhizodegradation, and biomass sequestration. Plant physiological responses further supported remediation success, with SPAD chlorophyll indices recovering significantly in nano-assisted treatments—indicating reduced oxidative stress and enhanced photosynthetic stability. Notably, hydrocarbon accumulation in plant tissues rose up to 2.3-fold in hybrid systems, underscoring biomass partitioning as a complementary removal pathway. Overall, the integrated nano-phytoremediation approach offers a robust, scalable, and mechanistically synergistic strategy for petroleum hydrocarbon remediation in aquatic environments, with broad implications for sustainable water treatment technologies.