Anti-malaria interventions that rely on insecticides can be compromised by insecticide-resistance alleles among malaria vectors. We examined frequency changes of resistance alleles at two loci, knockdown resistance (kdr) and acetylcholinesterase-1 (ace-1), which confer resistance to pyrethroids and DDT, and carbamates, respectively. A total of 7,059 Anopheles gambiae sensu stricto mosquitoes were analyzed from multiple sites across continental Equatorial Guinea. A subset of sites included samples collected pre-intervention (2007) and post-intervention (2009-2011). Both L1014S and L1014F resistance alleles were observed in almost all pre-intervention collections. In particular, L1014F was already at substantial frequencies in M form populations (17.6-74.6%), and at high frequencies (> 50%) in all but two S form populations. Comparison before and throughout anti-vector interventions showed drastic increases in L1014F, presumably caused by intensified selection pressure imposed by pyrethroids used in vector control efforts. In light of these findings, inclusion of other insecticide classes in any anti-vector intervention can be considered prudent.