This study aimed to investigated the prevalence and resistance pattern of different Citrobacter species phenotypically and genotypically to β-lactam and some most common antibiotics then evaluate the antibacterial activity of omega-3 extracted from flaxseed against isolates that harboring resistance genes. 19 Citrobacter isolates were isolated from100 stool and urine samples taken from patients attended to AL-Sadar Hospital during June-December 2016. Clinical samples were cultured on specific media, thereafter isolates were identified depending on morphological, biochemical characteristics and VITK-2. The results showed that the Citrobacter comprise 24% of isolated bacteria which divided into 11 (14.1%) were C. freundii, 5 (6.41%) C. kosaeri and C. farmeri were 3 (3.8%). The antagonistic activity was evaluated by observing a clear zone of inhibition growth, the results showed that all Citrobacter (100%) isolates were resistant to Ampicillin, cefoxitin and sensitive to Imipinim, also the isolates showed different degrees of resistant to β-lactam antibiotics initially. By confirmatory test the results observed 17/19 (89.4%) isolated were ESBL producers finally using PCR technique to detect bla–genes (blaCTX-M, OXA, SHV and Z). The results revealed that 14/17 (82.3) of potential ESBL producing Citrobacter were harbor one or more of ESBL genes they included 10 of C. freundii and 4 of C. koseri. The extraction of essential fatty acid semicarbazide (omega-3) from Linum usitatissium (Flax seed) were tested to evaluate their activity against resistant isolates, results explained broad spectrum antibacterial property of EFASC compounds against resistant bacteria. In conclusion, this study found increase prevalence of MDR Citrobacter spp as causative agents in clinical cases. Considering antibacterial activity of EFASC, it was observed highly activity against resistant pathogens deservedly, therefore attention must be paid to development their used as alternative antibiotics.