O'Higgins dropped to third in the table after their shock loss at Huachipato, who claimed a second consecutive victory to rise to 13th.
Huachipato had lost three in a row before last week's 1-0 win over Nublense and again tasted victory on Saturday thanks to Francisco Arrue's 25th-minute penalty.
Despite taking 11 shots to Huachipato's seven, O'Higgins could not find an equaliser and the hosts held on for the three points.
That left O'Higgins seven points adrift of Colo Colo, who had to survive a nervous few final minutes against Cobresal.
Juan Delgado's header from a corner was saved by Cobresal's Leonel Cuerdo but the hosts' midfielder followed up and stabbed out of the goalkeeper's hands and into the net.
Despite the visitors' protests, the goal stood and Colo Colo extended their lead with 26 minutes left when Felipe Flores won the ball from Cobresal defender Wladimir Herrera converted past Cuerdo.
Palestino moved within two points of O'Higgins in fourth spot with a 1-0 victory at Cobreloa on Sunday, while in the other late matches of the round Nublense defeated Rangers 1-0 and Everton trumped Santiago Wanderers 2-1.
