Postoperative endophthalmitis is a dreaded complication of intraocular surgery. Acute presentations need prompt management and good knowledge of differential diagnoses. In the last 10 years, progress in direct microbial detection and identification from intraocular samples included the use of blood culture systems and, more recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, improving the rate of bacterial identification. Whatever the method used, diagnostic sensitivity is better for vitreous samples than for aqueous humor samples. Besides, molecular biology techniques have further improved the identification rate of infectious agents in intraocular samples. They also provide faster results compared to culture-based techniques. Quantitative real-time PCR (qPCR) can also determine the bacterial load in intraocular samples. Several studies have shown that intraocular bacterial loads in endophthalmitis patients are usually high, which helps differentiating infection from contamination. The prognostic value of qPCR remains to be validated. Whole genome DNA sequencing technologies facilitate direct and sequencing of single DNA molecules. They have the potential to increase the rate of microbiological identification. Some antibiotic resistance markers (e.g., methicillin resistance in staphylococci and vancomycin resistance in enterococci) may be detected earlier using molecular techniques (usually real-time PCR tests). Early determination of the involved microorganism and their antibiotic resistances can help establishing an earlier therapeutic strategy.