<jats:title>Abstract</jats:title><jats:p>Human infections caused by microbial biofilm formation represent a growing major health threat. A new alkaloid, 3‐amino‐5‐(3‐hydroxybutan‐2‐yl)‐4‐methylphenol, was isolated from the corn grit culture of the endophytic isolate <jats:italic>Penicillium citrinum‐</jats:italic>314 associated with <jats:italic>Halocnemum strobilaceum</jats:italic>, a halophyte growing in the Egyptian marshes. The new alkaloid was identified by 1D, 2D‐NMR and HR‐ESI‐MS‐MS and given the trivial name halociline. The antioxidant, antimicrobial and antibiofilm activities were recorded. Furthermore, another known compound, 1,3,6‐trihydroxy‐7‐methoxy‐9<jats:italic>H</jats:italic>‐xanthen‐9‐one, was obtained in smaller amounts and revealed a non‐microbicidal 100 % reduction in biofilm formation, with an MBIC value of 62.5 μg/mL (228 μM) against <jats:italic>Pseudomonas aeruginosa</jats:italic> (Ferm‐BAM), a FRAP value of 447.941±37.876 mM/L as well as a marked safety profile against three cancer cell lines. Through <jats:italic>in silico</jats:italic> molecular docking study, in the binding sites of <jats:italic>Pseudomonas</jats:italic> enzymes, key ligand enzyme interactions were determined to support the <jats:italic>in vitro</jats:italic> results.