Despite the existence of an extended armamentarium of effective synthetic drugs to treat HIV, there is a continuing need for new potent and affordable drugs. Given the successful history of natural product based drug discovery, a library of close to one thousand plant and fungal extracts was screened for antiretroviral activity. A dichloromethane extract of the aerial parts of Daphne gnidium exhibited strong antiretroviral activity and absence of cytotoxicity. With the aid of HPLC-based activity profiling, the antiviral activity could be tracked to four daphnane derivatives, namely, daphnetoxin (1), gnidicin (2), gniditrin (3), and excoecariatoxin (4). Detailed anti-HIV profiling revealed that the pure compounds were active against multidrug-resistant viruses irrespective of their cellular tropism. Mode of action studies that narrowed the site of activity to viral entry events suggested a direct interference with the expression of the two main HIV co-receptors, CCR5 and CXCR4, at the cell surface by daphnetoxin (1).