The terrible intensification in global food demand predictably results in an increase in agricultural practices, with a focus on the usage of herbicides. Hence, a deeper understanding of the impact of this herbicide on aquatic ecosystems is required. The characterization of nano-emulsion of fluazifop-P-butyl (FL) and the toxic effects in both traditional and nanoforms on the growth, pigmentation and ultrastructure of the freshwater microalgae: Auxenochlorella pyrenoidosa and Raphidocelis subcapitata were studied. The prepared nano-emulsion has a particle size of 52-94 nm by transmission electron microscopy (TEM). Zeta potential recorded (38.5 mV) and polydispersity index recorded (0.394). Traditional fluazifop-P-butyl (TFL) in the case of A. pyrenoidosa exhibited median effective concentration (EC50) (10.909 ± 3.635 mg/L) (P= 0.235), while nano fluazifop-P-butyl (NFL) recorded EC50 (14.281 ± 6.251 mg/L) (P= 0.205). In the case of R. subcapitata, TFL recorded EC50 (1.174 ± 0.501 mg/L) (P= 0.105), while NFL exhibited EC50 (4.757 ± 1.755 mg/L) (P= 0.150). Exposure of R. subcapitata cells to 0.117 mg/L of TFL significantly decreased chlorophyll a level by (-)15.51% (P= 0.002) relative to control. Also, chlorophyll b content in A. pyrenoidosa cells significantly decreased upon treatment with 1.43 mg/L of NFL by (-)67.16% (P= 0.001). Carotenoid content in A. pyrenoidosa cells significantly increased after exposure to 1.09 mg/L of TFL by 108.88% (P= 0.001) compared to the control. Likewise, exposure of R. subcapitata cells to 0.117 mg/L of TFL elevated carotenoid levels by 200.57% (P < 0.001). Destroyed chloroplasts and compacted cell walls were observed through TEM when A. pyrenoidosa was exposed to 1.09 mg/L of TFL and 1.43 mg/L of NFL. Likewise, R. subcapitata cells were exposed to 0.117 mg/L of TFL and 0.476 mg/L of NFL. This study demonstrates that both conventional and nano-forms of FL endanger the integrity of the ecosystem and have adverse impacts on non-target organisms. Thus, it's crucial to follow biosafety protocols concerning non-target species before deciding to use this herbicide.