In nanoparticle biosynthesis, identification and/or characterization of extract and its biochemicals, playing a key role to control the morphology of nanoparticle, are important to minimize the synthesis steps, and thus to reduce environmental risks and cost. Ocimum sanctum plant leaves were subject to sequential solvent fractionation with different polarity (hexane, chloroform, n-butanol and water). In the steps, the four dried fractions were extracted and used for biogenic synthesis of gold nanoparticles. Gold nanoparticles of varying shape and size were synthesized according to the extraction solvents, i.e., chloroform fraction (circular discs with rough edges), hexane fraction (spherical nanoparticles smaller than 100 nm), aqueous fraction (large, anisotropic platelets with defined edges) and n-butanol fraction (Au aggregates). In second step, we identified the biochemicals in the extracts using gas chromatography−mass spectrometry (GC−MS). Methyl eugenol and β-caryophyllene were observed in the hexane fraction and eight known compounds, i.e., glycerol, phosphoric acid, succinic acid, tartaric acid, D-gluconic acid, and myo-inositol, 2-methoxy-4-vinylphenol and ferulic acid were identified in the aqueous fraction. Further, we checked that some identified biochemicals, i.e., ferulic acid, caryophyllene and 2 methoxy-4-vinylphenol synthesized microscale sheets, spherical particles and a branched nanostructure, respectively.